A number of organophosphorus (“OP”) compounds used by the agriculture industry and the military are highly toxic and thus hazardous to human health and harmful to the environment. For example, acetylcholinesterase-inhibiting OP compounds comprise the active ingredient of pesticides such as paraoxon as well as G-type nerve agents such as Sarin and Soman, etc., developed for chemical warfare. Thus, it is very important to be able to detoxify such OP compounds and to decontaminate surfaces and substances contaminated with these compounds.
One approach being investigated as a potential solution to this problem is enzyme-mediated detoxification. For example, a class of enzymes known as organophosphorus acid (“OPA”) anhydrolases (“OPAA”) (EC 3.1.8.2) can catalyze the hydrolysis of a variety of OP compounds, including pesticides and fluorinated “G-type” nerve agents, and such anhydrolases have been known to be produced via overexpression within the recombinant organism (see U.S. Pat. No. 5,928,927 to Cheng et al. incorporated herein by reference).
One family of organophosphorus compounds known as V-agents are particularly toxic and persistent. For example, ((Ethyl {2-[bis(propan-2-yl)amino]ethyl}sulfanyl) (methyl)phosphinate, known as VX, is very toxic to humans. The median lethal dose (LD50) for humans is estimated to be about 10 milligrams when contact is through skin. The estimated LCt50 for inhalation is estimated to be 30-50 mg min/m3. The native OPAA enzyme has been described to possess catalytic activity against various chemical nerve agents, but its activity against the particularly toxic and persistent V-agents such as VX (Ethyl ({2-[bis(propan-2-yl)amino]ethyl}sulfanylmethyl)phosphinate) and others is only marginally detectable, and therefore, not practically useful as a decontaminant or as a medical countermeasure for VX poisoning.
In addition to VX, other V-agents of interest include VR (Russian VX) N,N-diethyl-2-(methyl-(2-methylpropoxy)phosphoryl)sulfanylethanamine); CVX (Chinese VX) S-[2-(Diethylamino)ethyl] O-n-butyl methylphosphonothioate); and agent VM (S-[2-(Diethylamino)ethyl] O-ethyl methylphosphonothioate).
Methods of producing organophosphorus acid anhydrolases for detoxifying organophosphorus compounds are well known in the art.
U.S. Pat. No. 5,928,927 to Cheng et al. teaches expression and composition comprising wild-type organophosphorus acid anhydrolases (“OPAA-2”) from the Alteromonus sp. bacteria strain JD6.5.
U.S. 2013/0071394 to Troyer et al. teaches compositions and combinations containing an organophosphorus bioscavenger and a hyaluronan-degrading enzyme that can be used to treat or prevent organophosphorus poisoning, including nerve agent poisoning and pesticide poisoning. However, the bioscavenger that Troyer utilizes is also a wild-type OPAA.
U.S. Pat. No. 9,017,982 to Shah et al. teaches a non-wild-type organophosphorus acid anhydrolases having an amino acid substitution at position 212, such that the mutated OPAA may degrade (ethyl {2-[bis(propan-2-yl)amino]ethyl}sulfanyl) (methyl)phosphinate and other V-agents. However, the mutation occurs only at position 212 and the catalytic activity is only about 2-fold or less on VX, as compared to the wild-type OPAA. Therefore, new compounds and methods to effectively detoxify VX are needed.